Difference between revisions of "Part:BBa K1898250"
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We set up PCR to check if the DNA have the expected bands. We saw the bands at their expected size, which is ~2.1kb. | We set up PCR to check if the DNA have the expected bands. We saw the bands at their expected size, which is ~2.1kb. | ||
The DNA was then sent to sequencing. The sequencing results are unable to confirm the sequence from 854 to 895 bp. The four cutting sites are highlighted in red, promoter + rbs highlighted in light blue, GSR in orange, 10x Histidine-Tag in green, and terminator in dark blue. | The DNA was then sent to sequencing. The sequencing results are unable to confirm the sequence from 854 to 895 bp. The four cutting sites are highlighted in red, promoter + rbs highlighted in light blue, GSR in orange, 10x Histidine-Tag in green, and terminator in dark blue. | ||
Revision as of 15:22, 18 October 2016
Strong promoter + Strong RBS + GSR + 10x Histidine tag + Double terminator
BBa_K880005 consists of a strong promoter and strong RBS and was used to maximize protein production. This construct codes for GSR (glutathione reductase) and a 10x Histidine-tag. GSR is a catalyst for the conversion of glutathione disulfide to glutathione and 10x Histidine-tag is used for protein purification. BBa__B0015 is a double terminator used to stop transcription.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 804
Illegal BamHI site found at 1488 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 89
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1199
Illegal SapI.rc site found at 1607
Sequencing
We set up PCR to check if the DNA have the expected bands. We saw the bands at their expected size, which is ~2.1kb. The DNA was then sent to sequencing. The sequencing results are unable to confirm the sequence from 854 to 895 bp. The four cutting sites are highlighted in red, promoter + rbs highlighted in light blue, GSR in orange, 10x Histidine-Tag in green, and terminator in dark blue.
Sequence with vf2 primer:
Sequence with vr primer:
Although 854 to 895 bp is not confirmed in the two files provided here, this GSR DNA comes from BBa_K880005. From the sequencing file uploaded (https://parts.igem.org/Part:BBa_K1898200), 854 to 895 bp of the GSR gene is confirmed to be correct.